Bone is a complex system consisting of a carbonated apatitic calcium phosphate phase supported on a collagen-rich extracellular matrix. While the macroscopic properties of bone have been extensively investigated using different techniques in various biological systems, the early steps in bone tissue mineralization are not well understood. In an effort to characterize bone growth, numerous studies have been conducted to understand the chemical mechanisms involved in both de novo bone formation and bone remodeling. Despite this increased interest, an understanding of the processes governing early mineralization of bone is still incomplete. This proposal will use near-infrared Raman microspectroscopy and Raman imaging to study early mineralization in the mouse calvaria (the flat bones that comprise the top of the skull). The goal is to understand the steps leading to the deposition of new mineral and the chemical and physical transformations that the mineral undergoes during fetal and early postnatal development. Because Raman microspectroscopy is a relatively new tool in bone research, the work will begin with validation of protocols for Raman spectroscopy of bone tissue. The effects of standard specimen fLxing and embedding protocols and the potential Raman or fluorescence interference from a set of common histological stains will be examined. It is proposed that there will be few or no problems encountered beyond the known effects, such as protein cross-linking, of certain fixing protocols. If interferences due to the staining protocols occur, multivariate spectral and image processing will be used to overcome them. Because the progress of mineralization is accompanied by shifts in mineral spectral bands, the interpretation of small spectral band shifts will be put on a fawn foundation. In a subcontract to Central Michigan University, vibrational spectra (band positions and frequencies) of substituted hydroxyapatites will be computed by density functional theory and compared to Raman spectral measurements of model compounds (synthetic non-substituted and substituted hydroxyapatites) and on murine calvarial bone tissue. Systematic Raman imaging will be performed on calvarial sections harvested from normal mice just prior to the onset of mineralization (fetal day 15.5) until postnatal day 14. Raman imaging will be carried out at 1-day intervals during the prenatal period and at 2-day intervals during the postnatal period. Correlative immunohistological staining and Raman imaging will be used to understand the effects of bone sialoprotein and osteocalcin on the course of mineralization.